FIG. 1
Cul-1 associated with IκBα, β-catenin,…
FIG. 1
Cul-1 associated with IκBα, β-catenin, and SCF β TrCP is modified by Nedd8.…
FIG. 1Cul-1 associated with IκBα, β-catenin, and SCFβTrCP is modified by Nedd8. (A) Association of endogenous Nedd8 conjugated Cul-1 with IκBα or ectopically expressed βTrCP. Lanes 1 and 2, 293 cells were treated with 5 μM MG273 for 1 h and with TNF-α (10 ng/ml) for 10 min as indicated. Lysates were immunoprecipitated (IP) with anti-IκBα. Lanes 3 and 4, 293 cells were transfected with FLAG-tagged ΔF-βTrCP or WT-βTrCP, and lysates were immunoprecipitated with anti-FLAG resin. In all lanes, immune complexes were resolved by SDS-PAGE on 9% gels and immunoblotted with anti-Cul-1 and anti-Nedd8. (B) Ectopically expressed βTrCP associates with endogenous Cul-1 and Cul-1*. Lanes 1 and 2, 293 cells were transfected with FLAG-tagged WT-βTrCP and treated with TNF-α as indicated, and lysates were immunoprecipitated with anti-FLAG. Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-FLAG, anti-Cul1, or anti-Skp-1. Lane 3, cell lysate (30 μg) from nontransfected 293 cells analyzed as above. (C) Association of endogenous Cul-1 with β-catenin. Lanes 1 and 2, 293 cells were treated with or without 5 μM MG273 for 2 h as indicated. Lysates were immunoprecipitated with anti-β-catenin. Lanes 3 and 4, 293 cells were transfected with FLAG-tagged ΔF-βTrCP or WT-βTrCP, and lysates were immunoprecipitated with anti-FLAG resin. In all lanes, immune complexes were resolved by SDS-PAGE on 9% gels and immunoblotted with anti-Cul-1. Cul-1* = Nedd8-ligated Cul-1.
FIG. 2
Ubiquitination of IκBα requires the…
FIG. 2
Ubiquitination of IκBα requires the Nedd8 pathway. (A) Reconstitution of IκBα ubiquitination activity…
FIG. 2Ubiquitination of IκBα requires the Nedd8 pathway. (A) Reconstitution of IκBα ubiquitination activity in vitro. Ubiquitination assays were performed with HeLa FII (40 μg), ubiquitin (60 μM), an ATP-regenerating system, and recombinant 35S-labeled IκBα/p652 phosphorylated on S32 and S36 as described in the text. Into these reactions, HeLa FI (15 μg), UbcH5A (150 nM), Nedd8 (250 nM), and WT Ubc12 (150 nM) or Ubc12C111S (5 μM) was added as indicated. Following incubation at 37°C for 0 or 90 min, reactions were stopped by the addition of SDS sample buffer and resolved on an SDS–9% gel, and the reaction products were detected with a phosphorimager. (B) Ubc12C111S acts as a dominant negative inhibitor of IκBα ubiquitination. Reactions were performed as for panel A, using FII (40 μg), ubiquitin (60 μM), UbcH5A (150 nM), Nedd8 (1 μM), an ATP-regenerating system, and recombinant 35S-labeled IκBα/p652 phosphorylated on S32 and S36 as described in the text. Into these reactions, WT Ubc12 was added at 50 nM (lanes 1, 2 and 4), 500 nM (lane 5), or 1 μM (lane 6). Ubc12C111S (Ubc12 C-5; 5 μM) was added in lanes 3 to 6. Following incubation at 37°C for 0 or 90 min, reactions were stopped by the addition of SDS sample buffer and resolved on an SDS–9% gel, and the reaction products were detected with a phosphorimager.
FIG. 3
Identification of Nedd8 conjugation site…
FIG. 3
Identification of Nedd8 conjugation site in human Cul-1. (A) Alignment of Cul-1 amino…
FIG. 3Identification of Nedd8 conjugation site in human Cul-1. (A) Alignment of Cul-1 amino acid sequence with sequences of other cullins. The Nedd8 conjugation site identified in Cul-2 is indicated by an arrowhead. (B) K720R Cul-1 is defective in forming Nedd8 conjugates. FLAG-tagged WT Cul-1 (lanes 1 and 2) or K720R Cul-1 (lanes 3 and 4) was expressed in 293 cells and immunoprecipitated with FLAG resin. The immunoprecipitates were then incubated with recombinant Nedd8 (250 nM), Ubc12 (150 nM), and FII (20 μg) for either 0 (lanes 1 and 3) or 30 (lanes 2 and 4) min at 30°C. The reaction products were resolved by SDS-PAGE (7.5% gel) under reducing conditions and immunoblotted with the anti-FLAG antibody. Cul-1* = Nedd8–Cul-1.
FIG. 4
Nedd8 modification of Cul-1 stimulates…
FIG. 4
Nedd8 modification of Cul-1 stimulates ubiquitination activity of SCF β TrCP . 293…
FIG. 4Nedd8 modification of Cul-1 stimulates ubiquitination activity of SCFβTrCP. 293 cells were cotransfected with FLAG-tagged Cul-1 (WT or K720R) and Myc-βTrCP. (A) Anti-FLAG immune complexes from these lysates were immunoblotted with anti-FLAG to detect Cul-1, anti-Myc to detect βTrCP, and anti-Skp-1. Note the presence of Cul-1* in WT Cul-1-transfected cells only. (B) Immune complexes from WT Cul-1 plus WT βTrCP (lanes 1 to 5) or K720R Cul-1 plus WT βTrCP (lanes 6 to 10) were assayed for IκBα ubiquitination activity. Ubiquitination of WT (lanes 1 to 4 and 6 to 9) or S32/36A (lanes 5 and 10) IκBα (150 nM) was assayed in the presence of 100 nM E1, 60 μM ubiquitin, 0.5 μM ubiquitin aldehyde, 2.5 μM MG273, an ATP-regenerating system, and 1 μM microcystin LR. UbcH5a (150 nM), Cdc34 (150 nM), and the Nedd8 pathway (Nedd8 [250 nM], Ubc12 [150 nM], and APP-BP1/Uba3) were added as indicated.
FIG. 5
K720R Cul-1 affects the ubiquitination…
FIG. 5
K720R Cul-1 affects the ubiquitination activity of SCF β TrCP . 293 cells…
FIG. 5K720R Cul-1 affects the ubiquitination activity of SCFβTrCP. 293 cells were cotransfected as in Fig. 4 except that HA-Rbx1 was included. (A) Aliquots of anti-FLAG immune complexes were assayed for IκBα ubiquitination activity for 20 min at 37°C in the presence of 100 nM E1, 60 μM ubiquitin, 0.5 μM ubiquitin aldehyde, 2.5 μM MG273, an ATP-regenerating system, 1 μM microcystin LR, UbcH5a (150 nM), Cdc34 (150 nM), the Nedd8 pathway (Nedd8 [250 nM], Ubc12 [150 nM], 0.5 μl of affinity-purified APP-BP1/Uba3), and 150 nM IκBα/p652. Samples were analyzed by SDS-PAGE on 9% gels and quantified by phosphorimage (PI) analysis. Shown is conjugate formation plotted versus the amount of enzyme added. (B) Indicated amounts of the immune complexes assayed in panel A were separated by SDS-PAGE on 9% gels and immunoblotted with the indicated antisera. (C) Nedd8 conjugation to Cul-1 does not affect the Km for IκBα. Aliquots (5 μl [WT] or 10 μl [K720R]) of the anti-FLAG immune complexes shown in panel B were assayed for IκBα ubiquitination activity as in panel A, using IκBα/p652 ranging in concentration from 9.4 to 600 nM. Shown is conjugate formation plotted versus substrate concentration and fitted to the equation v = Vmax[S]/Km + [S].
FIG. 6
Ubiquitination of IκBα by SCF…
FIG. 6
Ubiquitination of IκBα by SCF β TrCP involves both the ubiquitin and Nedd8…
FIG. 6Ubiquitination of IκBα by SCFβTrCP involves both the ubiquitin and Nedd8 conjugation pathways. SCFβTrCP is composed of Skp-1, Cul-1, Rbx1, and the F-box protein, βTrCP. The ubiquitination activity of SCFβTrCP is potentiated when Cul-1 is modified by Nedd8. Nedd8 modification of Cul-1 occurs through a pathway which includes the heterodimeric Nedd8-activating enzyme, APP-BP1/Uba3, and the Nedd8-conjugating enzyme, Ubc12. The ubiquitin E2, Cdc34, is recruited to SCFβTrCP by interacting with Rbx1 and Cul-1. Both Cdc34 and UbcH5 have been implicated in ubiquitination of IκBα, but their precise relationship is unclear. The ubiquitin-E2s build a polyubiquitin chain on phosphorylated IκBα when IκBα is bound to βTrCP.
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